Age may be the greatest risk factor for human disease in the United States. While the macroscopic changes are easy to observe and quantify, many of the molecular and cellular mechanisms that underlie the aging process remain unclear. The budding yeast, Saccharomyces cerevisiae, is an excellent model for studying cellular aging because it is a highly tractable, unicellular eukaryote that also experiences a relatively short an finite replicative life span. Despite these advantages, genome- wide approaches for studying aging have been hampered by the extremely tedious assay for replicative life span. To overcome this obstacle, I propose to develop a novel assay for replicative life span that is amenable to high-throughput screening. The assay uses fluorescence-activated flow cytometry to determine the viability of a permanently labeled population of cells in culture. This assay is semi-automated, can be performed in a 96-well format and assays lifespan for thousands of cells, in contrast to the few cells that can be assayed by traditional assays. This novel assay wil allow the application of genome-wide screens for identifying important regulator of the aging process in the yeast model system. Using this assay, I will screen the entire yeast ORF overexpression library for genes that when overexpressed alter replicative life span. This overexpression screen is unique in that it will allow me to identify: 1) genes whose gene products become defective and/or insufficient with age, 2) genes whose gene products are negative regulators of life span, 3) genes that indirectly alter life span. In addition to performig the screen in typical rich growth media, I propose to assay the overexpression library in multiple media conditions, where yeast age in different metabolic states. Metabolism has been shown to play a significant role in aging, and the proposed work aims to identify metabolism-dependent and -independent regulators of life span using a series of unbiased, genome- wide screens. Knowledge of how aging occurs in a simple eukaryote may provide greater insight into how cellular aging occurs in general, as well as shed light on cellular functions that play important roles in aging and age-associated disease.